© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1933

EFFECTS OF SOME HEAVY METALS ON THE

SIZES OF THE MEDITERRANEAN MUSSEL

Mytilus galloprovincialis Lamarck, 1819

Levent Bat*, Funda Üstün, Oylum Gökkurt Baki and Fatih Şahin

Sinop University, Fisheries Faculty, Department of Hydrobiology, 57000 Sinop, Turkey

ABSTRACT

In the present study, several experiments were de-signed to evaluate the acute (96-h) and chronic (28-d) tox-icity of copper, lead and zinc in seawater on the survival of the Mediterranean mussel Mytilus galloprovincialis. The toxicity of these metals to mussels was evaluated by static and semistatic bioassays, calculating the LC50 values (le-thality concentrations for 50%) for different sizes of mus-sels. Survival decreased with increasing concentrations of Cu, Pb and Zn but the survival in seawater with dissolved Cu, Pb and Zn was higher in the presence of sediment than without sediment.

KEYWORDS: Bioassay, toxic, copper, lead, zinc, survival, sedi-ment, Mytilus galloprovincialis.

1 INTRODUCTION

The protection of marine habitats from damage due to the release of toxic metals can require toxicity test data on aquatic organisms, including bivalve molluscs. Mussels are valuable bioindicators, and commonly employed for the monitoring of metal pollution in the Black Sea [1-3], be-cause they are consumed by humans, have a wide geo-graphical distribution [4], and are an abundant component of the soft bottom marine benthic community. As filter-feeders, mussels have the capacity for accumulating met-als in their tissues, even low concentrations. Adults of mus-sels are sedentary and found attached to the substratum with byssal threads. They cannot escape from pollution. These facts make them particularly attractive for aquatic environmental monitoring studies. The Mediterranean mus-sel Mytilus galloprovincialis, in particular, offers many ad-vantages for toxicological research. Effects of heavy metal concentrations in seawater on molluscs are not just related to the metal concentrations in the surrounding environment, but also to the mollusc size. In the present study, there- fore, an attempt has been made to find out the size differ-

* Corresponding author

ences in sensitivity of the Mediterranean mussel M. gal-

loprovincialis, an ecologically and economically impor-tant mollusc species of the Black Sea coast, to the heavy metals copper, lead and zinc.

2 MATERIALS AND METHODS

2.1 Sample collection and experimental protocol

Animals were collected from rocky shores in inter-tidal zones by a scuba diver at Gazibey Rock (depth 15-22 m) known to be clean and to support a healthy population of M. galloprovincialis [3] (Fig. 1). Living specimens were transported immediately from the sampling sites to the Hy-drobiology Laboratory of Fisheries Faculty, Sinop Univer-sity, and they were kept in biological-filtered clean seawater into sediment-free plastic containers (20 x 20 x 25 cm). The clean seawater used for the experiments was collected from the mussels obtained. Aeration was continuously provided by gently bubbling air through disposable Pasteur pipettes connected via plastic tubes to an air pump. The animals were stored there for a period of 5-days acclimatization.

FIGURE 1 - Sampling area from Sinop coast of the Black Sea, Turkey.

Clean sediment was collected from the same area as

the mussels and washed through a 500-µm mesh sieve into a tank to remove any associated macrofauna, and then washed again at least 3 times with clean seawater before

© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1934

use in subsequent experiments. Collecting, storage, char-acterization and manipulation of sediments for toxicologi-cal testing is described by ASTM [5]. Sediment samples for total organic carbon analysis were dried at 105 ºC for 24 h. Five grams of dried sample were then treated with hydrochloric acid vapor overnight in a desiccating jar to convert any calcium carbonates to chlorides. Samples were then placed in a muffle furnace at 600 ºC for 4 h. The loss on ignition was taken as the organic carbon content of the sediment [6].

2.2 Bioassay procedure

Experiment 1: Toxicity of Cu, Pb and Zn to the different sizes of mussels for a for 96-h period

A stock solution of 1000 ppm of each metal was pre-pared by dissolving copper (CuSO4.5H2O), zinc (ZnCl2) and lead (Pb(NO3)2) in distilled water. Test solutions were prepared by diluting the stock solution with seawater. The mussels were exposed to 9 concentrations of copper, zinc and cadmium in seawater, as well as controls (uncontami-nated seawater), in setups with clean sediment. Each set-up consisted of three replicate containers of each of 9 concen-trations and 3 controls. Clean sediment was added to these containers to create an 8-cm deep layer. All containers were aerated in order to maintain the dissolved oxygen levels above 60% of the air saturation value [7, 8]. All containers were covered with black material to exclude direct light, except from directly above and 10 M. galloprovincialis

placed in each. No foods were supplied during the course of the experiment, nor were the test solutions changed.

Bioassays were conducted over a 96-h period after acclimatization of collecting individuals in the laboratory, initially with individuals comprising broad size categories (20-80 mm) in range finding tests. The mussels were exam-ined for survival as well as valve opening and closing ac-tivity. Subsequent experimentation was designed to com-pare the toxicities of Cu, Pb and Zn to small (20-39 mm), medium (40-59 mm) and large (60-79 mm) mussels (Ta-ble 1). Each container was examined daily. All dead ani-mals were removed and recorded. Dead mussels were those that did not exhibit shell closure in response to tactile stimulation. The LC50 was calculated by probit analysis [9]. Significant differences in toxicity between small, medium and large mussels were determined using the Duncan tests on percentage survival values [10].

TABLE 1 - Size (mm) and weight (g) of mussel used in this study.

Size Shell length (Min. -Max.)

Shell weight (Mean ± SE)

Whole soft parts (Mean ± SE)

Small 20-39 1.70±0.04 0.8±0.01 Medium 40-59 6.00±0.30 3.1±0.2 Large 60-79 12.75±0.35 7.8±0.4

Experiment 2: Toxicity of Cu, Pb and Zn to the mussels in the presence and absence of clean sediment for 96-h and 28-d periods

Ten mussels were randomly selected from the stocks and placed into each container. The animals were exposed

to the different concentrations of Cu, Pb and Zn in sea-water, with and without sediment, for both 4-days acute and 28-days chronic toxicity test. Size of mussels for hu-man consumption ranged between 50 and 80 mm [11]. Therefore, these sizes of the mussels were used in this second experiment. The tests were semi-static bioassays in which the exposure media were replaced every 96 h for 28-d bioassay. After each experiment, surviving individuals were counted, and these data were used in the calculation of the LC50, based on the lethal concentration at which 50% of the mussels did not survive. Each series consisted of 3 replicates with 10 animals. The test was run under a 16:8-h light:dark photoperiod. Temperature, oxygen, salinity and pH for each of the replicates used in all bioas-says were monitored daily to ensure that these were similar in all experiments.

3 RESULTS AND DISCUSSION

Parameters monitored during the toxicity tests indi-cated that the test conditions were consistent. Temperature was 15±3 ºC. The dissolved oxygen levels were above 60% of the air saturation value in all containers during the period of the bioassays. Salinity ranged between 17-18 ‰, pH ranged from 7.6 to 8.3. The changes in pH over a 28-d period were never >0.5 units in a given exposure. The total organic content of the sediment was 1.92 %±0.25.

No mortality was observed in all the controls, demon-strating that the holding facilities, seawater, control sedi-ment and handling techniques were acceptable for con-ducting the toxicity test, as required in the standard EPA/COE protocol, where mean survival should be less than 90%.

Experiment 1: Toxicity of Cu, Pb and Zn to the different sizes of mussels for a 96-h period

The LC50 values for each metal are given in Table 2. These data indicate that Cu is the most toxic, followed by Pb and Zn.

TABLE 2 - The 96-h LC50 values (mg.L1) for the Mediterranean mussel M. galloprovincialis exposed to Cu, Pb and Zn in seawater with clean sediment.

Size Cu

(Mean ± SE) Pb

(Mean ± SE) Zn

(Mean ± SE) Small 1.67±0.02 59±4.8 38±3.0 Medium 1.45±0.04 43±4.6 29±2.6 Large 1.18±0.07 39±3.6 18±2.6

Significant differences between small, medium and

large mussels were determined using the Duncan tests on percentage survival values. The results showed consis-tently higher survival rates for small mussels at 96-h, showing statistically significant differences (p<0.05). The 96-h LC50 values for the medium and larger mussels were lower than that of the smaller individuals. It is concluded that smaller individuals were more tolerant to metals than

© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1935

FIGURE 2 - Survival (%) of 50-80 mm sizes of the mussel Mytilus galloprovincialis exposed to Cu, Pb and Zn concentrations with and with-out clean sediment for 96 hours.

© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1936

FIGURE 3 - Survival (%) of 50-80 mm sizes of the mussel Mytilus galloprovincialis exposed to Cu, Pb and Zn concentrations with and with-out clean sediment for 28 days.

© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1937

those with medium and large sizes. Similarly, Daka and Ekweozor [12] studied the acute toxic effects of a crude oil to the mangrove oyster Crassostrea gasar. They found that the 96-h LC50 value for the larger oysters was lower than that of the smaller ones, implying that the latter were more tolerant to crude oil than the larger ones. It is ex-plained that smaller bivalves have the greater physiological vibrancy making them less susceptible to stress in general, and the upper limit of compensatory response was probably lower for the larger individuals leading to a faster failure of the detoxification systems [12].

Experiment 2: Toxicity of Cu, Pb and Zn to the mussels in the presence and absence of clean sediment for 96-h and 28-d periods

The results showed that presence or absence of sedi-ment in the ambient water has revealed that valve opening and closing activity is influenced by the metal concentra-tions. Clarke [13] pointed out that the closure of the valves in response to foreign substances in the environ-ment is a typical reaction of the mussels. The mussels are also able to sense the foreign substances [14]. Data in the present study indicate that mussels shut their valves con-tinuously at high metal concentrations in the ambient water, and open more than normal only when they have died. In this case, no response to close their valves was observed, even after prodding to stimulate them. Hietanen et al. [14] observed that, during the exposure, the mussel M. edulis reacted to the presence of Zn by not opening its valves. However, the mussels did not sense the presence of cad-mium at low concentrations [14]. This finding is similar to the present study. More than 90% of the mussels’ valves in the control containers were open. The normal valve opening activity of the mussels was observed when Cu, Pb and Zn concentrations were less than 2, 20 and 10 µg.g-1, respectively.

Survival of the mussels decreased with increasing concentration for each metal in seawater both with and without sediment for both 4 and 28 days (Figs. 2 and 3). When sediment was present, acute toxicity as measured by LC50 decreased for all metals. This is probably due to the adsorption of metals by the sediment causing a reduc-tion in the concentration of these metals in seawater im-mediately over the surface of the sediment [15]. However, surprisingly, even for 28 days, in the control M. gallo-

provincialis, survival was 100 % both without sediment and with sediment. This implies that metals were assimi-lated at a faster rate in the presence of sediment since water concentrations should have been lower under these conditions. This might have implied for toxicity [15]. They examined similar findings with the amphipod Coro-

phium volutator, and its conclusion was the same as that of McLusky and Phillips' [16] hypothesis that mortality was related not to the total amount of the metal accumu-lated but rather to the rate of its uptake. These findings agree with the present study and show the presence of sedi-ment in reducing metal toxicity to organisms [15]. Alterna-tively, due to filter-feeding mussels’, required for organic

matter, suitable sediment type, exposure to contaminated seawater without sediment may have been stressful factors [17]; somewhat surprisingly since stressed animals might be expected to grow less, or even lose weight [18]. This study clearly showed that the absence of sediment affected the M.

galloprovincialis increase sensitivity to metals.

4 CONCLUSIONS

- The Mediterranean mussel M. galloprovincialis is a sentinel species that is common throughout the Black Sea, and is easy to identify, collect for a bio-assay process, and appears to be a good bioindicator for metals.

- The M. galloprovincialis is widely used in marine environmental monitoring programs as well as labo-ratory and field toxicity studies.

- The Mediterranean mussel is ecologically and eco-nomically important and consumed by humans.

- There is an adverse relationship between survival and metal concentrations.

- The presence of sediment in the ambient seawater reduced the toxicity of metals to the Mediterranean mussel.

- Small sizes of the Mediterranean mussel are rela-tively resistant to the metals compared to medium or larger ones.

- The Mediterranean mussel is more sensitive to Cu than Pb and Zn, and Pb is less toxic to the mussels than Zn and Cu.

- The presence of sediment in metal-contaminated seawater increased the M. galloprovincialis survival rates.

REFERENCES

[1] Ünsal, M. and Beşiktepe, Ş. (1994). A preliminary study on the metal content of mussels, Mytilus galloprovincialis (Lmk.) in the Eastern Black Sea. Tr. J. Zoology, 18, 265-271.

[2] Bat, L., Gündoğdu, A., Öztürk, M. and Öztürk, M. (1999). Copper, zinc, lead and cadmium concentrations in the Mediter-ranean mussel Mytilus galloprovincialis Lamarck 1819 from Sinop coast of the Black Sea. Tr. J. Zoology, 23, 321-326.

[3] Bat, L., Üstün, F. and Gökkurt-Baki, O. (2012). Trace Ele-ment Concentrations in the Mediterranean Mussel Mytilus

galloprovincialis Lamarck, 1819 Caught from Sinop Coast of the Black Sea, Turkey. The Open Marine Biology Journal, 2012, 6, 1-5. DOI: 10.2174/1874450801206010001.

[4] Fish, J.D. and Fish, S.A. (1996). A student’s Guide to the Seashore. Second edition. Inst. of Bio. Sci, Univ. of Wales, Aberystwyth, England.

[5] American Society for Testing and Materials (1991). Standard guide for collecting storage, characterization and manipula-tion of sediments for toxicological testing. ASTM E 1391-90. American Society for Testing and Materials, Philadelphia, PA, pp. 1105-1119.

© by PSP Volume 22 – No 7. 2013 Fresenius Environmental Bulletin

1938

[6] Buchanan, J.B. (1984). Sediment analysis. In: N.A. Holme and A.D. McIntyre (Eds.), Methods for the Study of Marine

Benthos. Blackwell Sci. Publ., pp. 41-65.

[7] American Society for Testing and Materials (1990). Standard guide for conducting 10-day static sediment toxicity tests with marine and estuarine amphipods. ASTM E 1367-90. American Society for Testing and Materials, Philadelphia, PA, pp. 1-24.

[8] U.S. Environmental Protection Agency and U.S. Army Corps of Engineers (1991). Evaluation of dredged material pro-posed for ocean disposal. Testing manual. EPA-503/8-91/001, Washington, DC.

[9] Finney, D.J. (1971). Probit Analysis (3rd Edition). Cambridge University Press London, 256 pages.

[10] Zar, J.H. (1984). Biostatistical analysis. Second edition. Prentice Hall, Int., New Jersey.

[11] Aral, O. (1999). Growth of the Mediterranean mussel (Myti-

lus galloprovincialis Lam. 1819) on ropes in the Black Sea. Turkey. Tr. J. Vet. and Animal Sci. 23, 183-189.

[12] Daka, E.R. and Ekweozor, I.K.E. (2004). Effect of Size on the Acute Toxicity of Crude Oil to the Mangrove Oyster, Carasostrea gasar. Journal of Applied Sci. Environ. Man-

agement, 8 (2), 19-22.

[13] Clarke, G.L. (1947) Poisoning and recovery in barnacles and mussels. Biol. Bull., 92(1):73-91.

[14] Hietanen, B.,Sunila, I. and Kristofferson, R. (1988). Toxic ef-fects of zinc on the common mussel Mytilus edulis L. (Bival-via) in brackish water. I. Physiological and histological stud-ies. Ann. Zoll. Fennici, 25, 341-347.

[15] Bat, L. and Raffaelli, D. (1998). Sediment toxicity testing: A bioassay approach using the amphipod Corophium volutator and the polychaete Arenicola marina. J. exp. mar. Biol.

Ecol., 226, 217-239.

[16] McLusky, D.S. and C.N.K. Phillips (1975). Some effect of copper on the Polychaete Phyllodoce maculata. Est. and

Coast. Mar. Sci., 3, 103-108.

[17] Bat, L. (1996). Pollution effects on marine invertebrates. Ph.D. Dissertation, University of Aberdeen, Scotland, UK., 199 pages.

[18] Bat, L., Raffaelli, D.,Marr, I.L. (1998). The accumulation of copper, zinc and cadmium by the amphipod Corophium volu-

tator (Pallas). J. exp. mar. Biol. Ecol., 223 (2), 167-184.

Received: October 01, 2012 Accepted: January 17, 2013

CORRESPONDING AUTHOR

Levent Bat Sinop University Fisheries Faculty Department of Hydrobiology 57000 Sinop TURKEY Phone: +90 (368) 2876254 Fax: +90 (368) 2876255 E-mail: leventbat@gmail.com

FEB/ Vol 22/ No 7/ 2013 – pages 1933 - 1938